in vitro study of the antimicrobial activity of european propolis against paenibacillus larvae

Here, we tested the ability of propolis originating from the resins of these plants to inhibit the in vitro growth of Paenibacillus larvae , the organism that causes American foulbrood,

In vitro study of the antimicrobial activity of European

propolis against Paenibacillus larvae

Valery A ISIDOROV1,Krzysztof BUCZEK2,Grzegorz ZAMBROWSKI3,

Krzysztof MIASTKOWSKI1,Izabela SWIECICKA3,4

1

Forest Faculty, Bia łystok University of Technology, 17-200, Hajnówka, Poland

2

Department of Epizootiology and Clinic of Infectious Diseases, Faculty of Veterinary Medicine, University of Life

Sciences, Lublin, Poland

3

Department of Microbiology, University of Bialystok, Bialystok, Poland

4

Laboratory of Applied Microbiology, University of Bialystok, Bialystok, Poland Received 14 August 2016 – Revised 1 November 2016 – Accepted 22 November 2016

buds Here, we tested the ability of propolis originating from the resins of these plants to inhibit the in vitro growth of Paenibacillus larvae , the organism that causes American foulbrood, a fatal honeybee larval disease The study involved GC-MS analysis of extracts from nine samples of propolis gathered from the temperate climate zone of Europe The extracts showed noticeable differences in the content of flavonoids and other phenolic compounds Despite the differences in chemical composition, all tested extracts inhibited the growth of P larvae , with a

affect the strength of antimicrobial activity, but other phenolics, such as the phenylpropenoids hydroxycinnamyl sesquiterpenols, glycerides and benzoates also had an effect This is the first report on the comparative activity of different types of European propolis against P larvae.

Paenibacillus larvae / American foulbrood / Apis mellifera / propolis / antibacterial action

1 INTRODUCTION

American foulbrood (AFB), a disease of

hon-eybee larvae, has been known for more than

200 years Prior to the arrival into Europe and

North America of the parasitic mite Varroa

destructor , AFB was the most economically

im-portant disease of honeybees To date, AFB is the

most infectious and destructive disease of

honey-bee brood and is often fatal for honey-bee colonies

notifiable disease of honeybees and is subject to

decision regarding the method of combating (treating or destroying AFB colonies) is within the purview of the district veterinary surgeon The causative agent of this disease is the Gram-positive, spore-forming bacterium Paenibacillus larvae , which is highly infectious In some coun-tries, antibiotics or sulfathiazole are used to com-bat AFB; however, these drugs only suppress the clinical symptoms and cannot cure the disease, because they are not effective against the

this practice leads to serious consequences, such

as reduced honeybee vitality through the suppres-sion of the endogenous microflora in these bees, the almost inevitable pollution of beekeeping products with medicinal residues, and the appear-ance of bacterial resistappear-ance (Lodesani and Costa

Corresponding author: V Isidorov,

v.isidorov@pb.edu.pl

Manuscript editor: Stan Schneider

This article is published with open access at Springerlink.com

DOI: 10.1007/s13592-016-0485-z

legally banned in the European Union for use in

are classed as farming animals, whose products

cannot contain the residues of any drugs;

there-fore, this greatly limits the treatment possibilities

Practically the only nonmedicinal method that

is applied to sanitize infected colonies is the

so-called shook swarm method (proposed in 1769 by

Schirach), which consists of shaking adult bees onto

a new uninfected comb foundation and destroying

the old combs and other infected materials (Hansen

positive results in the treatment of infected but not

However, its application does not guarantee the

com-plete recovery of treated bee colonies Therefore, in

recent decades, considerable attention has been paid

to the development of alternative treatment methods,

based on the application of natural antibacterial

Propolis is a mixture of beeswax and resinous

material that is collected by honeybees from

var-ious plants In the mid-latitudes of the northern

hemisphere, the plant precursor of propolis is the

bud resins (exudates) of some arboreous trees, in

particular, different species of poplar, aspen, and

antimicro-bial activity of this natural antibiotic has been

attributed to phenolic substances: flavonoid

agly-cones, and phenolic and hydroxycinnamic acids

tends to vary to a great extent, depending on the

Recent publications have demonstrated that the

principal plant precursors of propolis from boreal

and temperate zones of the European continent are

the bud resins of black poplar (Populus nigra ),

downy birch (Betula pubescens ), and common

In a more recent investigation, taxonomical

markers of the resins from the buds of these trees

pop-lar bud resin is characterized by a high content of

phenols such as pentenyl (mostly, prenyl)

cinnamates, chalcones, and the unsubstituted B ring flavonoids: pinocembrin, pinostrobin, chrysin, galangin, pinobanksin, and their 3-substituted

birch and common aspen bud resins are

distin-g u i s h e d b y t h e p r e s e n c e o f s p e c i f i c phenylpropenoids, hydroxycinnamic acid esters

of sesquiterpene alcohols, and glycerol,

from the same colony often collect resins from more than one of these tree species, three main Btypes^ of European propolis can be distinguished

on the basis of the species-specific chemical com-position: poplar-, birch-, and aspen-type (Popova

the differences in the chemical composition are reflected in the antimicrobial activity of different Btypes^ of propolis; however, little is known about the effects of different types of European propolis

on P larvae The main aim of this communication was to compare the in vitro anti-P larvae action of three types of propolis from the temperate zone of the northern hemisphere in an attempt to relate the observed minimal inhibition concentration (MIC) values with the chemical composition of these bee products

2 MATERIALS AND METHODS 2.1 Chemicals and propolis

Pyridine, bis(trimethylsilyl)trifluoroacetamide

stan-dards were purchased from Sigma-Aldrich,

with diethyl ether (POCH SA, Gliwice, Poland) Two propolis samples (Pr-1 and Pr-2) were collected in the same apiary located in

Pr-3 originated from the forest-steppe in the

samples originated from the taiga zone: propolis

received from apiaries in the Vologda region

(59° 58′ N, 38° 31′ E and 59° 47′ N, 38° 38′ E).

Propolises Pr-1, Pr-2, and Pr-9 were collected

by the authors in the summer of 2015 To acquire

the material, a special net (mesh size of 1 mm) was

mounted just above the hive frames with the

brood After a time interval of 3 weeks, the net

became fully glued with pure propolis by the bees

The propolis was easily separated after cooling the

each) were gathered in the summer of 2015 by

apiarists in different countries Propolis was

har-vested by scraping it off the frames or by using a

plastic screens which were placed on the topmost

frames in the hive and leaved until the bees have

deposited propolis in the splits in the screen

2.2 Extract preparation and analyses

Two grams of ground propolis powder was

transferred to a 100 mL retort and extracted with

three 50 mL portions of diethyl ether for 24 h The

joint extracts were filtered through paper filter,

and the solvent was removed using a rotary

evap-orator The dry residue was used for chemical

analysis and antibacterial tests

About 5 mg of the residue was diluted with

mixture was sealed and heated for 30 min at 60 °C

to form trimethylsilyl (TMS) derivatives TMS

derivatives were analyzed using GC-MS on an

HP 6890 gas chromatograph fitted with an MSD

5973 mass selective detector (electron impact

source and quadrupole analyzer) from Agilent

Technologies (USA) This device was equipped

w i t h a n H P - 5 M S f u s e d s i l i c a c o l u m n

with electronic pressure control and a split/splitless

injector The latter was used at 250 °C in split

(1:50) mode The helium flow rate through the

components The analysis was carried out with

temperature programming from 50 to 310 °C at a

maintained for 15 min The MSD detector

acquisition parameters were as follows: the transfer line temperature was 280 °C, the MS source tem-perature was 230 °C, and the MS quad temtem-perature was 150 °C The electron impact mass spectra were obtained at an ionization energy of 70 eV The

After integration of the chromatogram, the frac-tion of each component in the total ion current (TIC) was calculated The precision of the method was studied by three replicate extractions and analyses The peak areas of the extract components obtained

by replicate analyses were used for the calculation of their relative standard deviation (RSD) values On average, RSD amounted to 2% for the main peaks (more than 10% of TIC), 6% for medium peaks (more than 1% of TIC), and 18% for peaks that

To identify the components, both mass spectral data and the calculated retention indices were used Mass spectrometric identification was car-ried out with an automatic system of GC-MS data processing supplied by the NIST 14 library (NIST/EPA/NIH Library of Electron Ionization Mass Spectra containing 276,259 standard mass spectra) and a home-made library of mass spectra The latter contains more than 1250 spectra of TMS derivatives prepared from commercial prep-arations of flavonoids, other phenolics, terpe-noids, aliphatic and aromatic acids, and alcohols The linear temperature programmed retention

and the TMS derivatives The measured values of the retention times were used to calculate the retention indices using the following equation:

IT ¼ 100 tðx−tnÞ= tðnþ1−tnÞ þ 100n

compound x and n- alkanes with the number of carbon atoms in the molecule n and n + 1,

2009,2014a,b; Isidorov2015) and presented in a home-made computer database containing more

compounds The identification was considered

reli-able if the results of the computer search of the mass

spectra library were confirmed by the experimental

home-made database values did not exceed ±5 u.i (the

highest quantity of intra-laboratorial deviation)

P larvae was isolated from honeybee larvae

and honey samples originating from apiaries in

dead larvae were aseptically removed from brood

combs and were crushed and suspended in 5 mL of

physiological saline (0.9% NaCl), followed by

shaking for 10 min at room temperature To isolate

bacteria from honey samples, about 10 mL of

honey was preheated at 45 °C, diluted with sterile

water in a ratio of 1:1, and centrifuged at 3000×g

for 30 min The resulting pellets were suspended in

crushed larvae and honey were centrifuged at

10,000 rcf for 5 min, and the resulting pellet was

All samples were preheated in a water bath for

10 min at 85 °C to eliminate vegetative cells and

to select the endospores After preparation of a

solu-tion was spread onto Columbia Blood Agar Base

Basingstoke, England) and incubated at 30 °C in

and that were transparent or slightly whitish, with

an elevated center and frayed edge, were initially

selected as P larvae and were cultivated on the

same medium and under the same conditions

men-tioned above to obtain pure cultures The bacilli

initially classified as P larvae were further inves-tigated by Gram staining, the catalase test

Gram-positive and catalase-negative bacilli that formed spiral forms in the Plagemann probe were

Mueller-Hinton broth (Oxoid) supplemented with glycerol

at a ratio of 1:1

2.4 16S rRNA gene sequencing Total DNA was prepared from overnight cul-tures of the isolates grown in a brain-heart infu-sion (BHI) broth using the protocol for Gram-positive bacteria with the DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany) and the Qiacube apparatus (Qiagen), according

to the instruction manuals The DNA concentra-tion and purity were checked using a NanoDrop

2000 spectrometer (Thermo Fisher Scientific Com., Waltham, USA) The partial 16S rRNA gene of the isolates was amplified using a pair of

Long Template PCR System (Roche Diagnostic GmbH, Mannheim, Germany) as follows: 94 °C for 3 min, 94 °C for 30 s, 50 °C for 45 s, and 68 °C for 7 min The 966-bp16S rRNA amplicons were cloned into pGEM-T Easy (Promega Corporation, Madison, USA) To determine the accuracy of the sequence, both strands of two clones were se-quenced using T7 and SP6 primers, in an ABI3500 automated sequencer (Applied Biosystems, Foster City, USA) For the compara-tive analyses of nucleotide and amino acid se-quences, database searches were performed using the BLAST program at the NCBI website (http://www.ncbi.nlm.nih.gov) This study confirmed the identification of isolates KB25, KB35, KB41, and KB55 as P larvae

The propolis extracts were tested against four Bwild^ P larvae isolates, as well as against the reference P larvae LMG 09820 strain (Belgian

Table I Paenibacillus larvae isolates used in the study.

Name of isolate Origin Country Region

KB25 Larvae Poland Lubelskie

KB35 Larvae Poland Lubelskie

KB41 Honey Poland Podkarpackie

KB55 Larvae Poland Podkarpackie

Coordinated Collections of Microorganisms) The

latter was isolated in 1906 from a foul brood of

honeybees by White as Bacillus larvae (White

finally named Paenibacillus larvae (Heyndrickx

were inoculated onto blood agar (Oxoid) and were

incubated for 48 h at 30 °C Then, bacteria were

reinoculated onto Mueller-Hinton broth and

0.4 at 600 nm, measured with a V-670

spectro-photometer (Jasco, Japan)

The propolis extracts were dissolved in DMSO at

Branson 2510 ultrasonic bath (Sigma), and filtered

filter (Carl Roth GmbH and Co, Karlsruhe,

Germany) To maintain appropriate nutrient

condi-tions, the extracts were aseptically diluted in

double-concentrated Mueller-Hinton broth using laboratory

tubes that contained an arithmetic dilution series

of 3 mL The propolis solutions used in the study

were as follows: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.2,

to observe whether the solvent alone caused

turbid-ity of the medium, we included a solvent control

The minimal inhibitory concentration (MIC) of

the extracts was assessed in the tube dilution test

in accordance with Clinical and Laboratory

Standard Institute (2011) protocols For the assay,

was added to each tube with the extract and was

incubated for 48 h at 37 °C The bacterial cultures

with different concentrations of propolises were

observed visually The lowest dose of the extract

with no growth of P larvae was regarded as the

MIC value All the tests were repeated four times

Cultures of P larvae in tubes with

Mueller-Hinton broth without propolis extracts were used

as a positive control Mueller-Hinton broth

sup-plemented with 10% DSMO arithmetically

dilut-ed ranging from 0.0025 to 5% was usdilut-ed as a

solvent control Similar growth of bacteria under

study in Mueller-Hinton broth without DMSO

(positive control) and in Mueller-Hinton broth

supplemented with 10% DSMO (solvent control)

was observed, which allowed to conclude that

DMSO itself does not affected P larvae growth

3 RESULTS The composition of diethyl ether extracts of propolis was very complex: on the chromatograms

of nine samples of propolis from five European

recorded, 141 of which belonged to different groups of aromatic compounds More comprehen-sive groups were formed by flavonoids and chalcones (53 unique chromatographic peaks) and by phenylpropenoids (63 unique peaks for cinnam ic acid derivatives) Apart from hydroxycinnamic acids and their benzyl and pentenyl esters (36 peaks), phenylpropenoids were also represented by the hydroxycinnamoyl esters

well as monoglycerides and diglycerides of p -coumaric, ferulic, and caffeic acids (17 peaks) Furthermore, all propolis samples contained

sever-al terpenoids in different amounts: the

(19 peaks) In general, the qualitative composition

of the samples under investigation corresponded well with previously published data on European

2014a) TableIIpresents the group composition of extracts in terms of their proportion of the total ion current (TIC) of the chromatogram

The specific features of the qualitative compo-sition of the propolis samples under investigation led to the conclusion that among the nine samples

Pr-9, were selectively collected by honeybees from a single plant source The propolis Pr-1 was almost a pure poplar-type propolis, Pr-5 was a birch type, and 9 was an aspen-type propolis Samples

Pr-2, Pr-3, and Pr-4 also belonged to the poplar-type; however, they contained a relatively small

glyc-erides, which are taxonomical markers of aspen

Pr-7 of Russian propolis were birch-type, but contained marked amounts (2.4 and 7.8% of TIC)

of aspen resin-derived phenylpropenoid glycer-ides Aspen-type propolis Pr-8 contained an ad-mixture (2.5% of TIC) of typical downy birch phenylpropenoid sesquiterpenols

Pr-1

Pr-3

Pr-4

Pr-5

Pr-6

Pr-7

Pr-8

Pr-9

Pr-2

0

100000

300000

500000

700000

900000

Time >

Abundance TIC: Pr_3KarSil.D\data.ms

0

100000

300000

500000

700000

900000

Time >

Abundance TIC: Pr_5KrySil.D\data.ms

0

100000

300000

500000

700000

900000

Time >

Abundance TIC: Pr_5KrySil.D\data.ms

0

50000

100000

200000

300000

400000

500000

Time >

Abundance TIC: Pr_11PalSil.D\data.ms

0

100000

300000

500000

700000

900000

Time >

Abundance TIC: Pr_4FerSil.D\data.ms

0

50000

100000

200000

300000

400000

500000

Time >

Abundance TIC: 120704_09.D\data.ms

0

50000

100000

200000

300000

400000

500000

600000

Time >

Abundance TIC: 120704_05.D\data.ms

100000

300000

500000

700000

900000

Time >

Abundance TIC: Pr_8TroSil.D\data.ms

50000

100000

200000

300000

400000

Time >

Abundance

TIC: 130419_07_P3.D\data.ms

Figure 1 GC-MS chromatograms of diethyl extracts of propolis samples Pr-1 to Pr-9.

Polish, Pr-1

Slovak, Pr-4

F

Hierarchical cluster analysis was performed

into three groups Samples Pr-2, Pr-3, Pr-4 form one group harvested from poplar buds Pure poplar-type sample Pr-1 belong to the same group, showing a difference from other samples of this

bud resins Samples Pr-5, Pr-6, and Pr-7 were grouped as another group harvested presumably from birch buds A slight difference shows sample Pr-5 which contains the least admixture of aspen resins The third group was formed of samples

Pr-8 and Pr-9 harvested from aspen buds, which are considerably different from other samples in

anti-P larvae activity (see below)

To test the antimicrobial activity of the propolis extracts, we used the tube dilution assay The

09820 strain of P larvae were tested The minimal inhibitory concentrations of the propolis extracts

the Polish poplar-type propolis Pr-1, which showed

In contrast, the Latvian pure aspen-type propolis Pr-9 showed lower antimicrobial activity (from

4 DISCUSSION

It is relevant to ascertain how the chemical composition of different propolis types influences their activity against P larvae As can be observed

differences in their composition, all tested extracts significantly inhibited the growth of the Polish isolates and the reference strain of P larvae with

The investigated poplar-type propolis (P-1 to P-4) showed practically identical effects on the pathogen, which is not surprising considering that their qualitative composition was very similar Moreover, the quantitative compositions of the biologically active phenols in P-1 to P-4 were similar if the lipophilic wax components were excluded Therefore, the slightly higher activity

assay with KB25, KB35, and KB41 isolates might

be due to the lower (compared to propolis samples

Polish, Pr-1

Slovak, Pr-4

F

Pr-2 to Pr-4) relative content of Bneutral^ wax

components such as saturated fatty acids and their

esters with saturated aliphatic alcohols (6.3% of

TIC), alkanes, and alkenes (7.7% of TIC), which

do not influence the biological activity of propo-lis Notably, samples Pr-1 and Pr-2 were collected

Figure 2 Hierarchical cluster analysis of propolis samples based on their chemical composition.

Table III Minimal inhibitory concentration of different extracts for P larvae.

Propolis MIC (μg mL −1 ) for P larvae strains

Poplar type

Birch type

Aspen type

at the same time by honeybees inhabiting

neigh-boring hives It appears that one of the two bee

colonies, for unknown reasons, added more waxes

to the collected poplar bud resins

The high anti-P larvae activity of the poplar-type

and birch-type propolis can be attributed to the high

content of flavonoids, which is consistent with

differ-ence in the composition of the flavonoid fraction of

these two types of propolis was notable The

prod-ucts derived from poplar bud resin contained large

amounts of dihydroflavonols (pinobanksin and its

3-substituted derivatives) and the flavanones

pinocembrin and pinostrobin, which were

contrary, only small amounts of the main birch-bud

flavanone sakuranetin was present in poplar-type

propolis Sakuranetin and another flavanone,

homoeriodictyol, as well as the flavone

pectolinaringenin can be considered as taxonomic

markers of downy birch (B pubescens ) on par with

phenylpropenoid sesquiterpenols (Isidorov et al

2014b,2016) These flavonoids possess

spectrum of biological activity of propolis from the

boreal zone of Europe, including anti-P larvae

action

Although they only contained trace amounts of

flavonoids, the aspen-type samples of propolis were

be attributed to other phenols, i.e., hydroxycinnamyl

glycerides This hypothesis needs experimental

con-firmation; phenylpropenoid glycerides have

demon-strated antitumor, antiproliferative, and estrogenic

properties have not been previously investigated

Notably, aspen-type Pr-8 propolis, containing an

admixture of birch bud-derived phenylpropenoid

sesquiterpenols, was slightly more active than the

pure aspen-type sample Pr-9

These findings firstly assume that not only

fla-vonoids but also different phenylpropenoids (such

as phenylpropenoid glycerides, the main phenolics

of aspen-type propolis) are also responsible for the

antibacterial activity of propolis from the boreal

zone Secondly, the higher anti-P larvae activity

com-ponents derived from birch bud resin, might pro-vide epro-vidence of a synergistic interaction among different compounds in propolis (Mihai et al

with respect to protection against different

study the interaction effects among phenols of different classes of propolis extracts

Finally, a comparison of the results of investiga-tions dealing with the in vitro anti-P larvae action

of essential oils and propolis confirmed the higher activity of propolis For example, essential oils from four Citrus species demonstrated MIC values

relatively high MIC values (from 50 to

Lavandula officinalis , Cinnamomum zeylanicum , Mentha piperita , Pimpinella anisum , and Foeniculum vulgaris Therefore, the application

of propolis for nonmedicinal AFB treatment ap-pears to be more promising

ACKNOWLEDGEMENTS

The project was supported by the Grant of National Science Center (Poland) 2014/13/B/NZ7/02280.

Contributions VI —propolis collection and GC-MS anal-ysis; KB—P larvae isolation and identification; GZ and IS—microbiological investigations; KM—participation in the design of the data

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Etude in vitro de l ’activité anti-microbienne de propolis

d ’origine européenne vis-à-vis de Paenibacillus larvae Paenibacillus larvae / loque américaine / Apis mellifera / propolis / action antibactérienne